Final exam - MB409 - May 10, 2002 Name _____Key_____

All questions are worth 5 points

1. What are the 3 primary evolutionary branches of life?

Archaea, Bacteria, and Eukarya

2. Esther Angert used PCR to amplify ssu-rRNA sequences from samples of Epulopiscium picked one-at-a-time from fish guts. Why did she then make fluorescent oligos specific to these ssu-rRNA sequence and use them to probe fish gut contents by in situ hybridization?

To show that the ssu-rRNA sequences she cloned & sequenced actually came from Epulopiscium.

3. Sap-sucking aphids with bacterial symbionts (B. aphidicola) get essential amino-acids and vitamins from the bacteria. What do the bacteria get from this symbiosis?

They get a place to live free of competition, all the nutrients they need for survival, and an efficient route for transmission to the aphids offspring.

4. Describe the local environment in Octopus Spring where the "pink filaments" live.

It is a ca. 85C outflow spring from the main "source" pool of Octopus. narrow and very shallow, the outflow is filled with course sinter gravel, which is covered with the pink filaments. The water is mineral-laden, neutral to slightly alkaline in pH, with low concentrations of both hydrogen and oxygen.

5. What is the purpose of bootstrapping?

To assess the reliability of the branches of a phylogentic tree.

6. When Robert Huber was attempting to grow the pink filaments organism, how did he evaluate his enrichment cultures? In other words, how did he know which cultures were were working and which were not?

He probed his enrichment cultures (FISH) using probes specific to the pinl filamentous organism sequence EM17.

7. In attempt to obtain ssu-rRNA sequences from primitive eukaryotes in Obsidian Pool (a.k.a. "Jim's Black Pool"), Sue Barns used primers that were specific for: (circle one)

A. Eukaryotes only
B. Archaea only
C. Bacteria only
D. Eukaryotes and Archaea
E. Eukaryotes and Bacteria
F. Archaea and Bacteria
G. all three phylogenetic groups

8. How did Philip Hugenholtz sort the 300 clones he obtained, using universal and bacterial-specific rRNA primers with DNA samples from Obsidian Pool, into groups of related sequences?

By RFLP analysis. He PCR amplified the ssu-rDNA from each clone and digested it with a couple of frequently-cutting restriction enzymes and ran the products on a gel. Clones with the same pattern were lumped together into a group for further analysis.

9. Philip Hugenholtz also probed ssu-rRNA, amplified from Obsidian Pool DNA using universal primers, with bacterial and archaeal-specific probes. What did he show in this experiment?

That the large majority of organisms in the Obsidian Pool sediment were Bacteria, not Archaea as would be expected.

10. What is a "chimeric" ssu-rRNA sequence?

An ssu-rRNA sequence that contains sequences from more than one source. Chimeras are artifacts of PCR amplification from DNA from environmental samples.

11. How did Ed DeLong count the relative numbers of Archaea, Bacteria, and Eukarya in samples of marine picoplankton?

By FISH using probes specific for each of the 3 main phylogenetic groups.

12. When Rudolf Amann probed samples of wastewater sludge with fluorescently-labeled phylogenetic probes, how was he able to distinguish 7 different phylogenetic groups using only 3 different probes?

Because each organism could bind to any possible combination of the 3 probes, and each probe is labeled with a different fluorescent tag, there are 7 possible color combinations, each of which represents a unique phylogenetic group.

13. In their experiments attempting to detect Yersinia pestis in the dental pulp of children that presumably died of plague in Middle Age France, what did Michel Drancourt use as a positive control to make sure he was successful in obtaining DNA from his samples of sufficient purity and quantity for PCR?

He used the beta-globin gene as a positive control. Because this gene is from the host, it should be present in all the samples of DNA and should give a PCR product if the DNA samples were successfully purified and clean.

14. Again, in their experiments detecting Yersinia pestis DNA in the dental pulp of children that died of plague in Middle Age France, why could they use dental pulp from unerupted teeth, and why is that better than other parts of the skeletal remains?

He could use dental pulp because it contains blood, and Yersina is a systemic infection. Dental pulp from unerupted teeth is best because it is encased in bone and well-preserved, minimizing decay and contamination from decay organisms.

15. In their experiments showing that stingless bees preserved in amber since the lower Miocene or upper Eocene contained (in life) symbiotic Bacillus species, what experiment did Raúl Cano use to argue that the sequences obtained by PCR were not from contamination?

He separated the insects into abdomen, thorax, and head samples, and showed that only the abdomens, where the symbionts are primarily located, yeilded PCR products.

16. In their experiments showing that Bacillus spores trapped in salt for 250 million years could be revived, what experiment did Russell Vreeland use to argue that the cultures obtained were not from experimental contamination?

He did a series of control experiments asssessing the sterility of his equipment, and mock experiments of salt samples without inclusions, to show that it is very unlikely that the growth came from experimental contamination.

17. In the paper by Birger Rasmussen, the fossil remains of filamentous prokaryotes in a "massive sulfide deposit" in western Australia were described. The massive sulfide deposit is the fossilized remains of the environment these organisms lived in. What was that environment?

A deep-sea hydrothrmal veent field.

18. David McKay and his colleagues had several lines of evidence from Martian meteorite ALH84001 for previous life on Mars. The polyaromatic hydrocarbon (PAH) data has since been largely dismissed. The carbonate "fossils" are visually striking, but not convincing because of their size and size distribution, and the uncertainty in their time and place of origin. One other line of evidence, however, is much better and so far has withstood the test of time. Describe this evidence.

Magnetite beads found in the meteorite are remarkably similar to those found in magnetotactic Bacteria. The only known way to form such beads is biologically.

19. You have graduated with your degree in Microbiology, and are now working on your Ph.D. in the Department of Microbiology at Allamance Community & Metropolitan Extension (ACME) University. Your new lab works on an organism isolated from scorched coyote fur. However, you show that 3 very similar, but not quite identical, ssu-rRNA sequences can be amplified from this culture, even though it's supposed to be pure. How could a pure culture be represented by more than one ssu-rRNA sequence? How would you go about determining whether you have a pure culture or not?

There are several possible approaches, but one way would be to design fluorescent oligonucleotide probes spcific to each of the ssu-rRNA variants, and use these probes in FISH experiments to make sure they all hydridize to all of the cells of the culture. You could also streak the organism out, and sequence ssu-rRNA clones from individual colonies (each colony comes from a single cell) to show that all 3 sequences are present in all single colonies.

20. You have graduated with your degree in Microbiology, and have landed a job at ACME Scientific, Inc. Your first task is to help with an ongoing problem they are trying to solve for a client that has problem with rapid corrosion in some factory pipework. The pipes, when opened, seem to have an unusual biofilm in the places where corrosion is happening, and samples of this biofilm contain lots of rod-shaped Gram-negative Bacteria when examined microscopically. However, attempts to culture this organism have failed. How would you identify the organism that seems to be causing this problem?

You could extract DNA from a sample of the pipe biofilm, amplify ssu-rDNA by PCr and clone the products, then sequence a number of clones. Fluorescent probes specific to whatever sequences you get would be used in FISH experiments to show which sequence(s) is present in the biofilm.